Lactic acid is a three carbon carboxylic acid with the molecular formula C3H6O3 (MW=90.08), containing a hydroxyl group adjacent to the carboxyl group: α-hydroxy acid or 2-hydroxypropanoic acid. Lactic acid is soluble in water or ethanol, hygroscopic, and recognized as GRAS (Generally Regarded As Safe) by the U.S. FDA (Narayanan et al., Electronic J. Biotechnol. 7:167-179, 2004). In solution, lactic acid can lose a proton from the carboxyl group, producing the lactate ion (CH3CH(OH)COO−).
The applications of lactic acid in the food and other chemical industries are diverse; it is used as an acidulant/flavoring/pH buffering agent or inhibitor of bacterial spoilage in a wide variety of processed foods. For example, a technical grade lactic acid is used as an acidulant in vegetable industries. Lactic acid in food products usually serves either as a pH regulator or as a flavoring agent. A related compound that is made from lactic acid and used as a food preservative is calcium stearoyl-2-lactylate. Lactic acid is also used as a humectant or moisturizer in food processing and some cosmetics, and as a mordant, a chemical that helps fabrics accept dyes in textiles. Moreover, lactic acid is used in the pharmaceutical industry as a starting material for the synthesis of substances of pharmaceutical importance. It is also utilized in the manufacturing of lacquers and inks. In addition, lactic acid is an important component of making industrially valuable chemicals such as polylactic acid, a biodegradable plastic, ethyl lactate (C5H10O3; MW=118.13) and acrylic acid (C3H4O2; 72.06).
The global market for lactic acid has been estimated to reach 329,000 metric tons by 2015 (Global Industry Analysts Inc, January 2011). The current market price for 88% food grade lactic acid is $1,400-1,600/metric ton. Furthermore, the major lactic acid manufacturers are: PURAC; Myriant; Archer Daniels Midland Company; CSM N.V.; Galactic S.A; Henan Jindan Lactic Acid Co. Ltd.; Musashino Chemical Laboratory Ltd.; and Musashino Chemical (China) Co. Ltd. The recent announcements of plant expansions and building of new development-scale plants for producing lactic acid and/or polymer intermediates by major U.S. companies, such as Cargill, Chronopol, A.E. Staley, and Archer Daniels Midland (ADM), attest to this potential. Major international manufacturers of fermentative lactic acid include Purac (Netherlands), Galactic (Belgium), and several Chinese companies. In late 1997, Cargill joined forces with Dow Chemical and established a Cargill-Dow PLA polymer venture, NatureWorks LLC, which exists today as a stand-alone company. NatureWorks LLC has constructed a major lactic acid facility in Blair, Nebr., which has the capacity of producing 180,000 metric tons of lactic acid per year, and it began operating in late 2002. The growing lactic acid market is and will in future be driven largely by rising oil prices, stringent government regulations and greater consumer interest toward the use of greener products (Global Industry Analysts Inc, January 2011).
Lactic acid can be produced via chemical synthesis or biological fermentation. In chemical synthesis (Narayanan et al., Electronic J. Biotechnol. 7:167-179, 2004), hydrogen cyanide (HCN) is first added to acetaldehyde (CH3CHO) in presence of a catalyst to produce lactonitrile (CH3CHOHCN). This reaction occurs in liquid phase at high atmospheric pressures. The crude lactonitrile is recovered and purified by distillation. Lactonitrile is then hydrolyzed to lactic acid, either by concentrated hydrochloric acid (HCl) or by sulfuric acid (H2SO4) to produce the corresponding ammonium salt and lactic acid (CH3CHOHCOOH). Lactic acid is then esterified with methanol (CH3OH) to produce methyl lactate (CH3CHOHCOOCH3) which is removed and purified by distillation and hydrolyzed by water under acid conditions to produce lactic acid and methanol, according to the Eqs. 1-4:CH3CHO+HCN→CH3CHOHCN  Eq. 1CH3CHOHCN+H2O+½H2SO4→CH3CHOHCOOH+½(NH4)2SO4  Eq. 2CH3CHOHCOOH+CH3OH→CH3CHOHCOOCH3+H2O  Eq. 3CH3CHOHCOOCH3+H2O→CH3CHOHCOOH+CH3OH  Eq. 4
The chemical synthesis route produces a racemic mixture of D(−)-lactic acid and L(+)-lactic acid, which are not suitable for some specific applications like synthesis of polylactic acid (PLA), one of the most promising end-use markets for lactic acid. Hence, about 90% of current commercial lactic acid is obtained via biological fermentation of sugars (Hofvendahl and Hahn-Hägerdal, Enzyme Microbiol. Technol. 26: 87-107, 2000; Zhou et al. Biotechnol. Lett. 28:663-670, 2006). Biological fermentation of sugars produces stereo-specific D(−)-lactic acid or L(+)-lactic acid depending on the strains used. For example, the microorganisms Lactobacillus, Bacillus, Rhizopus, Streptococcus, and Enterococcus produce L(+)-lactic acid while microorganisms such as Leuconostoc and Lactobacillus vulgaricus produce D(−)-lactic acid (U.S. Pat. No. 7,682,814).
L(+)-lactic acid is the preferred component for many food and industrial applications. L(+)-lactic acid is currently produced via biological fermentation utilizing lactic acid bacteria (LAB) or fungi such as Rhizopus (Maas et al., Appl. Microbiol. Biotechnol. 72:861-868, 2006). Some recombinant yeast strains have also been demonstrated to enhance lactic acid production from various carbon feedstocks (U.S. Pat. No. 7,326,550). However, as both yeast and fungal strains have low yield and productivity of lactic acid, compared to LAB, they are generally not preferred for industrial production of lactic acid. In addition, the mycelial morphology of fungal strains can result in increased viscosity of the fermentation medium and can cause blockages around the impellers (Sun et al., Biochem. Eng. J. 3:87-90, 1999).
Most industries use recombinant Lactobacillus sp. for lactic acid production. However, it is known that most Lactobacillus strains are fastidious that require expensive nutritional components and complex organic substances to support their growth and metabolisms as the LAB cannot generate most of the growth regulatory factors on their own. The desirable characteristics of industrial microorganisms are their ability to rapidly ferment inexpensive feedstocks, requiring minimal amount of nitrogenous substances, and produce high yields of stereo-specific lactic acid with low amounts of byproducts. Furthermore, as the purity of food-grade lactic acid supplied by the industries is on average between 80% and 90%, production of a pharmaceutical-grade lactic acid with purity higher than 90% will increase the cost of purification of lactic acid, which in turn will reflect on its price (John et al., Appl. Microbiol. Biotechnol. 74:544-534, 2007). Accordingly, there is a need for a method of lactic acid production with high titer, yield and volumetric productivity utilizing a less fastidious microorganism capable of growing on simple and inexpensive fermentation medium.
Recent studies and research developments, as presented in the referenced patents and incorporated in their entirety below, describe the use of wild-type and recombinant microorganisms for lactic acid production. However, to date, no patent literature has described the use of Enterococcus faecalis for lactic acid production. In published research literature, E. faecalis RKY1 was used to produce up to 93 g/L lactic acid at 1.7-3.2 g/L/h and pH 7.0 on wood hydrolyzate containing up to 100 g/l glucose equivalents and supplemented with 15 g/l yeast extract (Wee et al., Biotechnol. Lett. 26:71-74, 2004). Another Enterococcus species, E. flavescens, produced 28 g lactic acid/L at pH 5.5 on cheese whey as carbon source and corn steep liquor as nitrogen source under controlled anaerobic conditions after 30 h of fermentation (Agarwal et al., Biotechnol. Lett. 30:631-635, 2008).
U.S. Pat. No. 4,698,303, granted on Oct. 6, 1987, discloses a method of producing lactic acid by Lactobacillus casei at a cell mass concentration of 60 g/L, using continuous fermentation employing medium pretreatment, cell-recycle fermentation, fermentation broth acidification, and lactic acid separation. Enzymatic digest of whey was used as a nitrogen base in the culture medium. The fermentation was carried out at pH 5.0-6.5 and temperature 40° C.-45° C. utilizing lactose as a carbon source at a feed rate of 0.25 fermentor volumes/h. The yield and rate of lactate production was above 90% and 12 g/L/h, respectively. However, the specific productivity was very low (0.2 g lactate/g dry cell/h).
U.S. Pat. No. 7,326,550, granted on Feb. 5, 2008, describes the production of lactic acid utilizing recombinant yeast strains. According to the patent, yeast strains such as Kluyveromyces lactis, Torulaspora delbrueckii, Saccharomyces sp. and Zygosacchoromyces bailii, lacking ethanol production ability or with reduced ethanol production ability and/or reduced pyruvate dehydrogenase and pyruvate decarboxylase activities, were transformed with a copy of the gene encoding lactic dehydrogenase (LDH), functionally linked with a promoter sequence of the yeasts or with a heterologous expression by overexpressing a lactate promoter. The recombinant yeasts produced 0.052-0.757 g lactic acid/g glucose in a medium containing 1% yeast extract, 2% peptone and 10% glucose. A maximum concentration of 109 g/L of free lactic acid was achieved with a Kluveromyces yeast carrying LDH gene, designated PMI/C1[pEPL2], however, the final yield and productivity of lactic acid decreased to 0.59 g/g glucose and 0.795 g/L/h, respectively. The fermentation medium was enriched with expensive complex substrates like yeast extract and peptone that represent significant obstacles to industrial scale up.
In U.S. Pat. Pub. No. 2010/0190222, published on Jul. 29, 2010, a method of producing and separating lactic acid in a culture medium from fermentation culture is described. Lactic acid was produced by recombinant yeast strains using glucose at 10% as a carbon source for 72 h. The final lactic acid yield of 26% and productivity of 0.36 g/L/h are very low for commercial utilization.
In U.S. Pat. No. 7,682,814, granted on Mar. 23, 2010, a method of producing lactic acid at high concentration and high yield was described using the strain Lactobacillus paracasei CJLA0310 KCCM-10542. This strain was shown to produce 179 g/L of lactic acid from 180 g/L of glucose (yield of 99.5%) with an average productivity of 3.85 g/L/h a high cell density culture (OD600 of 24). Although the lactic acid yield and titer are high, this organism was cultivated in a nutrient-rich fermentation medium containing large amounts of complex organic substances such as yeast extract (15 g/L) and peptone (10 g/L) which is believed to increase the cost of lactic acid production and purification from the complex medium (Narayanan et al., Electronic J. Biotechnol. 7:167-179, 2004).
U.S. Pat. No. 2011/0171703, published on Jul. 14, 2011, discloses a recombinant bacterium, Escherichia coli, transformed with a gene encoding one NAD-dependent lactate dehydrogenase and one NAD-independent lactate oxidoreductase. The recombinant E. coli produced 97 g lactic acid/L in a culture medium containing 120 g glucose/L and 30 g yeast extract/L at pH 7.5 in 18 h. Drawbacks of this method are the relatively low lactic acid yield (80%) and the use of high amounts of yeast extract as a nitrogen base which leads to increased production and purification costs.